Solid-phase nucleic acid isolation

Details Solid-phase nucleic acid isolation Solid-phase nucleic acid isolation

2000-02-07

2003-09-09

Fredman, Jeffrey (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S270000, C435S259000, C536S127000, C536S023100

Reexamination Certificate

active

06617105

ABSTRACT:

The present invention relates to the isolation of nucleic acid, and especially to a method for isolating DNA from cells which combines a solid phase cell isolation step with a solid phase DNA isolation step.
The isolation of nucleic acid is an important step in many biochemical and diagnostic procedures. For example, the separation of nucleic acids from the complex mixtures in which they are often found is frequently necessary before other studies and procedures eg. detection, cloning, sequencing, amplification, hybridisation, cDNA synthesis etc. can be undertaken; the presence of large amounts of cellular or other contaminating material eg. proteins or carbohydrates, in such complex mixtures often impedes many of the reactions and techniques used in molecular biology. In addition, DNA may contaminate RNA preparations and vice versa. Thus, methods for the isolation of nucleic acids from complex mixtures such as cells, tissues etc. are demanded, not only from the preparative point of view, but also in the many methods in use today which rely on the identification of DNA or RNA eg. diagnosis of microbial infections, forensic science, tissue and blood typing, detection of genetic variations etc.
The use of DNA or RNA identification is now widely accepted as a means of distinguishing between different cells or cell types or between variants of the same cell type containing DNA mutations. Thus, HLA typing, which is more commonly carried out by identification of characteristic surface antigens using antibodies, may alternatively be effected by identification of the DNA coding for such antigens. Microbial infection or contamination may be identified by nucleic acid analysis to detect the target organism, rather than relying on detecting characterising features of the cells of the microorganisms eg. by morphological or biochemical. Genetic variations may be identified by similar means.
In general, DNA or RNA is identified by hybridisation to one or more oligonucleotides under conditions of stringency sufficient to ensure a low level of non specific binding. Commonly, the hybridising nucleotides are used in pairs as primers in the various forms of in vitro amplification now available, primarily the polymerase chain reaction (PCR), but also the Ligase Amplification Reaction (LAR), the Self-Sustained Sequence Replication (3SR) and the Q-beta replicase amplification system. After amplification the DNA may be further characterised by sequencing, eg. by the Sanger method. Amplification and sequencing may be combined.
As mentioned above, all methods generally require an initial nucleic acid isolation step, to separate the nucleic acid from materials eg. protein which may interfere in the hybridisation and amplification techniques which are used.
A range of methods are known for the isolation of nucleic acids, but generally speaking, these rely on a complex series of extraction and washing steps and are time consuming and laborious to perform.
Classical methods for the isolation of nucleic acids from complex starting materials such as blood or blood products or tissues involves lysis of the biological material by a detergent or chaotrope, possibly in the presence of protein degrading enzymes, followed by several extractions with organic solvents eg. phenol and/or chloroform, ethanol precipitation, centrifugations and dialysis of the nucleic acids. Not only are such methods cumbersome and time consuming to perform, but the relatively large number of steps required increases the risk of degradation, sample loss or cross-contamination of samples where several samples are simultaneously processed.
Improvements in methods for isolating nucleic acids are thus continually being sought, and more recently, other methods have been proposed which rely upon the use of a solid phase. In U.S. Pat. No. 5,234,809, for example, is described a method where nucleic acids are bound to a solid phase in the form of silica particles, in the presence of a chaotropic agent such as a guanidinium salt, and thereby separated from the remainder of the sample. WO 91/12079 describes a method whereby nucleic acid is trapped on the surface of a solid phase by precipitation. Generally speaking, alcohols and salts are used as precipitants.
Whilst such methods speed up the nucleic acid separation process, a need still exists for methods which are quick and simple to perform, which enable good yields to be obtained without losses, and in particular which are readily amenable to isolating nucleic acids from cells in mixtures or environments where they may be present at low concentrations, as a preparative first step in isolating nucleic acids from target cells in nucleic-acid based cell detection procedures. The present invention addresses this need. In particular, whilst hybridisation-based techniques such as PCR and other nucleic acid-based methods for detecting microorganisms allow high sensitivity detection of cells in samples, sample preparation ie. the concentration of the target cells and nucleic acid purification, are crucial factors in achieving the high sensitivity and reproducibility of the method. At present, cells are commonly first isolated from the sample by filtration, centrifugation or affinity binding to antibodies attached to a solid phase. After cell concentration in this manner, the DNA is then purified from the concentrated cells, often by classical phenol/chloroform extraction methods as discussed above, with their attendant disadvantages.
We now propose a novel approach to this problem which integrates cell isolation and nucleic acid purification in a single “step”, by using the same solid phase for both cell adsorption and nucleic acid purification. This is achieved by binding the cells to a solid support as a first step. The same solid support is then used under conditions that lyse the bound cells, and then which enable the nucleic acid to bind to the support.
In this manner nucleic acid may be isolated from a sample in a form suitable for amplification or other downstream processes, by a simple and quick to perform procedure which may take less than 45 minutes.
In one aspect, the present invention thus provides a method of isolating nucleic acid from a sample of cells, said method comprising:
(a) binding cells in said sample to a solid support to isolate cells from the sample;
(b) lysing the isolated cells; and
(c) binding nucleic acid released from said lysed cells to said same solid support.
The nucleic acid may be DNA, RNA or any naturally occurring or synthetic modification thereof, and combinations thereof. Preferably however the nucleic acid will be DNA, which may be single or double stranded or in any other form, eg. linear or circular.
The term “cell” is used herein to include all prokaryotic (including archaebacteria) and eukaryotic cells and other viable entities such as viruses and mycoplasmas, and sub-cellular components such as organelles. Representative “cells” thus include all types of mammalian and non-mammalian animal cells, plant cells, protoplasts, bacteria, protozoa and viruses.
The sample may thus be any material containing nucleic acid within such cells, including for example foods and allied products, clinical and environmental samples. Thus, the sample may be a biological sample, which may contain any viral or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts and organelles. Such biological material may thus comprise all types of mammalian and non-mammalian animal cells, plant cells, algae including blue-green algae, fungi, bacteria, protozoa etc. Representative samples thus include whole blood and blood-derived products such as plasma or buffy coat, urine, faeces, cerebrospinal fluid or any other body fluids, tissues, cell cultures, cell suspensions etc., and also environmental samples such as soil, water, or food samples.
The sample may also include relatively pure or partially purified starting materials, such as semi-pure preparations obtained by other cell separation processes.
Binding of the cells to the so

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